The aim of this study is to assess the influence of cardiopulmonary coupling (CPC) based on RCMSE on the prediction of complications and death in patients with acute type A aortic dissection (ATAAD).The cardiopulmonary system may be nonlinearly regulated, and its coupling relationship with postoperative risk stratification in ATAAD patients has not been studied.This study was a single-center, prospective cohort study (ChiCTR1800018319). We enrolled 39 patients with ATAAD. The outcomes were in-hospital complications and all-cause readmission or death at 2 years.p < 0.001). When CPC-RCMSE was used to predict all-cause readmission or death at 2 years, the AUC was 0.731 (p < 0.05). After adjusting for age, sex, ventilator support (days), and special care time (days), CPC-RCMSE remained an independent predictor of in-hospital complications in patients with ATAAD [adjusted OR: 0.8 ].Of the 39 participants, 16 (41.0%) developed complications in the hospital, and 15 (38.5%) died or were readmitted to the hospital during the two-year follow-up. When CPC-RCMSE was used to predict in-hospital complications in ATAAD patients, the AUC was 0.853 (CPC-RCMSE was an independent predictor of in-hospital complications and all-cause readmission or death in patients with ATAAD. CPC is Since the cardiopulmonary system may be regulated in a nonlinear way, a new coupling analysis technique is needed. In recent years, the multiscale entropy (MSE) algorithm has gained massive attention, especially in the cardiovascular and physiological fields . Norris No known research has focused on exploring the relationship between the definition of CPC based on MSE and the complications and deaths of patients with ATAAD. Therefore, we propose the following hypotheses: the cardiopulmonary coupling index based on refined composite multiscale entropy (CPC-RCMSE) can be used for successful risk stratification in ATAAD patients.2.2.1.This was a prospective cohort study involving all patients after aortic dissection at the First Affiliated Hospital of Wenzhou Medical University from September 2018 through September 2020 (ChiCTR1800018319). The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University.The inclusion criteria were as follows: (1) patients aged 18–65 years; (2) patients who met the diagnostic criteria for acute type A aortic dissection and underwent surgery; and (3) patients or family members who voluntarily participated in the trial and signed informed consent. The exclusion criteria were as follows: (1) patients with chest and back deformity (including appearance and organic); (2) pacing patients; (3) patients with an ECG signal quality not up to standard; (4) patients in the terminal stage of chronic wasting disease; (5) patients in persistent coma; and (6) pregnant or nursing women.2.2.Electrocardiograph signals and respiratory waveforms were collected for more than 4 h on the first day after returning to the cardiac care unit. The CPC-RCMSE formula was described previously .2.3.The main end point was the occurrence of death, acute liver failure, acute renal failure, or ventilator-associated pneumonia composite end events in the hospital. The secondary endpoint was all-cause readmission or death at 2 years.2.3.1.t test or the Mann‒Whitney U test, and groups of categorical variables were compared using the χ2 test or Fisher's exact test.Continuous variables are expressed as the mean ± standard deviation (SD). Categorical variables are expressed as the number (percentage). Groups of continuous variables were compared using Student's p < 0.05 was statistically significant.Univariate logistic regression analysis was used to investigate the independent risk factors for the compound endpoint of complications in the hospital in patients with ATAAD. All variables significantly associated with in-hospital mortality were candidate variables in the stepwise multivariate analysis. Based on the results of the multivariate logistic regression analysis, we explored whether CPC-RCMSE is an independent predictor of complications in the hospital. A receiver operating characteristic (ROC) curve for CPC-RCMSE was generated. Survival curves were described by the Kaplan‒Meier method and compared by the log-rank test. Statistical analyses were performed by SPSS 25.0 statistical software  and the R language tool. In all analyses, 3.3.1.In this study, patients with aortic dissection were selected. The results are presented in 3.1.1.p < 0.05].The univariate logistic analysis results are shown in 3.2.p < 0.001). When CPC-RCMSE was used to predict all-cause readmission or death at 2 years, the AUC was 0.731 (p < 0.05).As shown in 3.3.p < 0.0001).As shown in 4.p < 0.001), indicating that CPC-RCMSE had a good ability to predict the occurrence of complications in the hospital. When CPC complexity was ≤21.2, the sensitivity was 75%, and the specificity was 86.7%. When CPC complexity was used to predict readmission and death, the AUC was 0.731 (p < 0.05), also indicating good differentiation ability. The results of multivariate analysis indicate the clinical prediction and risk stratification value of CPC-RCMSE.In reviewing the literature, no data were found on the association between CPC-RCMSE and prognosis in ATAAD patients who have undergone surgery. Our findings highlight the prognostic importance for ATAAD. As expected, patients in the highest quintile of CPC-RCMSE had better outcomes. When CPC-RCMSE was used to predict the occurrence of postoperative complications in ATAAD patients, the AUC was 0.853 .The results of our study indicated that CPC-RCMSE was an independent risk factor for ATAAD. ATAAD patients were divided into three groups according to their CPC-RCMSE . The Kaplan‒Meier curves showed significant survival outcomes for all-cause readmission or death at 2 years by CPC-RCMSE score. In patients with lower CPC-RCMSE scores, survival was worse (Additionally, we found that CPC-RCMSE was a strong independent predictor associated with in-hospital complications in our study population. When CPC complexity increased by 1 unit, the risk of hospital complications decreased by 20%. Clinical health care workers can be more targeted in meeting patient needs based on CPC-RCMSE scores to improve medical efficiency, shorten hospital stay, reduce care unit costs, and improve patient hospitalization outcomes.5.CPC-RCMSE is an independent predictor of in-hospital complications in patients with ATAAD. CPC-RCMSE should be used more widely and routinely in the risk stratification of ATAAD patients.


In radiotherapy, especially when treating children, minimising exposure of healthy tissue can prevent the development of adverse outcomes, including second cancers. In this study we propose a validated Monte Carlo framework to evaluate the complete patient exposure during paediatric brain cancer treatment.Organ doses were calculated for treatment of a diffuse midline glioma (50.4 Gy with 1.8 Gy per fraction) on a 5-year-old anthropomorphic phantom with 3D-conformal radiotherapy, intensity modulated radiotherapy (IMRT), volumetric modulated arc therapy (VMAT) and intensity modulated pencil beam scanning (PBS) proton therapy. Doses from computed tomography (CT) for planning and on-board imaging for positioning (kV-cone beam CT and X-ray imaging) accounted for the estimate of the exposure of the patient including imaging therapeutic dose. For dose calculations we used validated Monte Carlo-based tools , while lifetime attributable risk (LAR) was estimated from dose-response relationships for cancer induction, proposed by Schneider et al.μSv (testes) and 1.3 mSv (thyroid) for CBCT and 0.03 μSv (testes) and 48 μSv (scapula) for X-ray. Adding imaging dose from CT and daily CBCT to the therapeutic demonstrated an important contribution of imaging to the overall radiation burden in the course of treatment, which is subsequently used to predict the LAR, for selected organs.Out-of-field organ dose equivalent data of proton therapy are lower, with doses between 0.6 mSv (testes) and 120 mSv (thyroid), when compared to photon therapy revealing the highest out-of-field doses for IMRT ranging between 43 mSv (testes) and 575 mSv (thyroid). Dose delivered by CT ranged between 0.01 mSv (testes) and 72 mSv (scapula) while a single imaging positioning ranged between 2 The complete patient exposure during paediatric brain cancer treatment was estimated by combining the results from different Monte Carlo-based dosimetry tools, showing that proton therapy allows significant reduction of the out-of-field doses and secondary cancer risk in selected organs. Complete patient exposure during paediatric brain cancer treatment is estimated by combining different dosimetry tools.Imaging dose significantly contributes to the out-of-field doses in proton therapy while its contribution is proportionally much lower for photon treatments.Proton therapy allows to considerably decrease the out-of-field doses and thus risk of secondary cancer when compared to photon therapy.1Improvements of radiotherapy procedures have had a major impact on survival of paediatric patients. While benefits to the patient largely outweigh risks associated with the therapeutic use of ionising radiation, the late effects of exposure are particularly important to understand for children with high probability of tumour control.Recent large cohort studies of children exposed to low doses from computerised tomography (CT) scans have shown increased risks of leukaemia and brain tumours –5. Very In this context, the HARMONIC project , 14 aimsPaediatric patients undergoing radiotherapy are exposed to ionising radiation, as a consequence of the treatment, but also from complementing imaging procedures. Experimental dosimetry studies have been performed extensively within the European Radiation Dosimetry Group (EURADOS) WG9, using paediatric anthropomorphic phantoms during photon therapy –17 and mUntil now, the imaging dose during radiotherapy was generally considered negligible in clinical practice because of its low magnitude compared to the therapeutic dose given at the treated volume. Nevertheless, the use of on-board imaging (OBI) for accurate patient positioning has become even more frequent for advanced radiotherapy, such as proton therapy. Therefore, sufficient attention should be given to the dose delivered to the patient by imaging procedures. Moreover, doses from therapeutic exposures should be complemented with imaging doses to have a complete picture of the absorbed dose distribution.Within HARMONIC, a tool for calculating the dose from imaging procedures during radiotherapy has been further developed . FurtherPrevious studies have used the absorbed dose , 28 or hA Monte Carlo study on fetal dose during brain radiotherapy considered the biological effects of neutrons by estimating the quality factor provided in ICRP Publication 60 (ICRP 1991) for proton therapy. This enabled a fair comparison between proton and photon therapy demonstrating a 10-fold reduction in the fetal dose between PBS proton therapy, and 3D-CRT . Others 22.1Aiming to simulate a realistic treatment of a brain tumour, a clinically applied treatment plan was transferred to the conditions of the experiment. A 7-year-old female patient was selected with a diffuse midline glioma (WHO grade IV). The patient received a combined radiotherapy and chemotherapy after R3 resection. The concerned patient was enrolled in the prospective registry study ‘KiProReg’  after consent from her legal guardians. This study was approved by the local ethics committee.Dprescribed =50.4Gy(RBE) with 1.8Gy(RBE) per fraction was prescribed to the initial planning target volume (PTV), which was located in the cerebellum and had a volume of 195.2 cm3. The proton treatment plan consisted of two ipsilateral oblique fields and a contralateral oblique field , Inc.) representing a 5-year-old child (type 705D). A median dose of ield see .>95% regarding Dprescribed. More details can be found in a recently published paper . All photon irradiations for this study were done with a Varian TrueBeam STx LINAC operating with a flattening filter at a nominal energy of 6 MV. The linac was equipped with a Varian Millennium 120 multileaf collimator. The same dose of 50.4Gy with 1.8Gy per fraction was prescribed to the initial PTV. The 3D-CRT plan used two lateral fields with beam angles 90° and 270°. The IMRT plan consisted of five coplanar and isocentrical fields with beam angles of 70°, 125°, 180°, 235° and 280°, respectively. VMAT was planned using two 360° isocentric rotations. The plans were optimised with the photon optimisation algorithm PO . The plans were iteratively optimised over several steps using the constraint V7Gy=4% for the eye and V40Gy=5% and V25Gy=5%, for the left and right cochlea respectively, and V98%[PTV]ed paper .2.2In order to evaluate doses delivered by X-ray based imaging systems during the course of either proton or photon therapy, Monte Carlo simulations computed the imaging absorbed dose distributions on the paediatric anthropomorphic phantom for all imaging exams that the actual treatment would have required, namely the CT exam used for planning and the OBI exams used for positioning during treatment. In practice, the proton therapy centre uses daily X-ray imaging protocol while the photon therapy centre uses daily kV-CBCT (kilo-voltage cone beam computed tomography) protocol for all radiotherapy techniques.2.2.1For planning exams, CT protocols vary very little within the same treatment centre. Nevertheless, the scan length and the X-ray tube current are often dependent on the patient morphology and pathology. Thus, we used as reference protocol the one actually delivered to the paediatric patient treated at the West German Proton Therapy Centre Essen (WPE) on its Philips Big Bore CT scanner : 120 kVp, single fixed filter, 12 mm collimation, 210 mA, 287 mm of scan length and an exposure time of 31.9 s.2.2.2To depict the daily OBI exams performed during a radiotherapy treatment on a TrueBeam (Varian) we selected the kV-CBCT ‘head low dose’ protocol. It corresponds to an irradiation on a partial anteroposterior arc of 200° performed at 100 kVp, with the full-fan filter  and 146 mAs (20 mA and 20 ms per projection and 364 projections), using 22.2 cm × 16.6 cm field size at source-axis distance (SAD). This exam is repeated at each treatment session and for the studied clinical case this corresponds to 28 times .2.2.3To portray the daily OBI practice, we used the WPE X-ray protocol optimised for position verification of tumours with localisation in the head of children with the proton gantry positioned at 0°. It consists of making a first image using the X-ray tube A, on the same direction as the treatment beam at 90 kVp, 12 mA and 100 ms ; as well as a second image with X-ray tube B, oriented at 270° from the treatment beam direction at 90 kVp, 32 mA and 100 ms . This exam is repeated at each treatment session and for the studied clinical case, this corresponds to 28 times.2.3The whole-body absorbed dose distributions presented have all been computed with general-purpose radiation transport Monte Carlo codes. In all cases, the DICOM-CT image of the anthropomorphic 5-yearold CIRS phantom was used for the Monte Carlo radiation transport. The validations of these simulations were done by comparison of the Monte Carlo-computed doses with the experimental values obtained by detectors, such as thermoluminescent detectors (TLDs) and bubble detectors, inserted in the CIRS phantom. These validations have been already published, as well as the detailed description of the simulations and experiments , 25. Fi2.3.1PRIMO (version 1.0.64.1814) , 36, a M2.3.2The Geant4 –46 wrap-2.3.3The Monte Carlo framework for computing imaging absorbed doses is based on an in-house modified version of PENELOPE 2006 that introduced parallelisation and the possibility to use voxelised geometries [previously described in ]. CalculExperimental Monte Carlo model validation for both systems are presented in 2.4The CIRS phantom contains 180 organ-specific inserts and allowed to estimate the dose equivalent for 22 organs by averaging the calculated data from organ-specific locations. For radiotherapy we calculated the dose equivalent per organ considering a total target dose of 50.4Gy(RBE). For proton therapy, an RBE of 1.1 was considered and an absorbed dose of 45.8Gy was used for the normalization of out-of-field organ dose. For photon therapy the total absorbed target dose was 50.4Gy. For imaging, the dose equivalent per organ was calculated from a single imaging procedure for CT, kV-CBCT and X-ray. Then, we summed the dose equivalent per organ for the different imaging procedures by assuming the following: i) a single planning CT scan (1*CT) and, ii) a daily OBI (28*kV-CBCT or 28*X-ray). Finally, to get an estimate of the total dose equivalent per organ, during the entire radiotherapy treatment, the dose equivalent from radiotherapy and imaging was summed for each organ.In the plots that follow, the error bars represent the spread on the calculated average dose equivalent per organ and not the uncertainties. The number of points in an organ varies among organs as described by the manufacturer . Standar2.5In this study we applied the carcinogenesis model, previously published , to estiD is the average dose equivalent, at the respective organ location, as computed within our study (units mSv) and β(EAR) is referring to the initial slope, which is the slope of the dose-response curve at low dose for each site. These are tabulated in Table 1 of Schneider et al.  in equation 1 describes the age variation of EAR and depends on the age of exposure e and the attained age a in years:The function eγ and aγ for a Japanese population and for different sites are taken from Table 1 in Schneider et al.  for the patient, which is the EAR integrated from a = e to the life expectancy amax. The determination of LAR was done as described by Kellerer et al.   is the 33.1In The out-of-field dose equivalent during proton therapy is in all cases lower than photon therapy techniques and ranges from 120mSv in the thyroid down to 0.6mSv in testes. The difference to photon techniques becomes larger, the further away from the target. For example, the out-of-field dose equivalent ratio between IMRT and proton therapy ranges from 4.8 in thyroid up to 74 in testes.3.2μSv.Doses during imaging procedures were calculated using Monte Carlo-based software. μSv (testes) and 1.3mSv (thyroid). For X-ray the dose equivalent ranges between 0.02μSv (testes) and 56μSv (scapula). Organs in the thorax region spreading over long distances in the coronal plane, such as sternum, ribs and lungs, demonstrate large spread of computed dose equivalent, indicating a large dose gradient.In When considering a daily imaging procedure, the total imaging dose equivalent per organ is plotted in 3.3For photon techniques the imaging dose equivalent is lower than the therapeutic dose equivalent. Still, close to the field, the contribution of imaging dose equivalent can be important Figure 63.4A final comparison between radiotherapy techniques is made by considering the additional dose equivalent from imaging. Here we use the daily CBCT as the most conservative approach, as it resulted in the most elevated imaging dose equivalent, and compare the total dose equivalent for the different radiotherapy techniques in The ratio of photon to proton dose equivalent increases when computed further away from the target. In the thyroid the ratio is around 4 for all photon techniques, while for the bladder the ratio is 36, 58 and 17 when comparing 3D-CRT, IMRT and VMAT, respectively to PBS proton therapy.3.5In 4The current Monte Carlo-based framework allows to study the complete patient exposure during paediatric brain cancer and the potential subsequent risks for secondary cancer induction from available dose-response models. The accuracy of the presented simulations was experimentally shown in previous publications.Stray radiation in proton therapy is dominated by neutrons, therefore we considered the higher RBE, by applying the Q-factor of neutrons in the Monte Carlo software and reported on dose equivalent per organ, as described in De Saint-Hubert et al. . For pho3) compared to the previous study [65cm3  coy [65cm3 . Secondly [65cm3 , 53. FinOne needs to be cautious when favouring one technique to another as this comparison is only for the specific out-of-field organs considered for which measurements were performed. Indeed, the published cancer risks represent only an under-estimation of the probable overall risk of secondary cancer, which should include sarcoma, non-malignant brain tumours (e.g. meningioma), carcinoma for organs located in-field and haematopoetic tumours for the overall risk ratio. It must also be pointed out that the cancer risks for organs in the medium and high dose range can behave quite differently with regard to the various irradiation techniques than in the low dose range , 55. ThiImaging dose equivalent was most pronounced for CT and more than one order of magnitude higher when compared to CBCT. CT dose equivalent data were, however, higher when compared to the data obtained within the EPI-CT study . For exaμSv at far distances while in the study from Hälg et al. . The extra X-ray procedures are only done at the first radiotherapy treatment but we have calculated the impact of this extra dose. As expected the dose equivalent from X-ray procedure increased and this was on average by a factor of 3, when compared to a single angle (gantry 0°). However, this was only for the first treatment fraction and the impact on the total dose equivalent was within 15%. Therefore, we did not report on the extra dose from different angles. Moreover, X-ray doses are so low that the contribution to the total dose equivalent will be very limited. For this reason, the total dose equivalent applied during radiotherapy was calculated for daily OBI with kV-CBCT, which would result in the most conservative estimate of the dose equivalent per organ and associated risk.P = 0.009). In our study the risk was reduced by a factor of 6 when studying protons versus IMRT, which could be related to the fact that we did not calculate the risk to all organs, because of missing dose-response relationship for some organs, as well as the fact that we considered only organs far out-of-field. Moreover, the study of Xiang et al. (P = 0.001). This was not observed in the current study, where the risk estimations show a reduced risk for 3D-CRT compared to IMRT. One possible explanation is that this work only analyses the cancer risk for organs in the low-dose volume. However, in the low dose volume, the increased scatter and leakage dose with IMRT contributes to an increased cancer risk for these organs. For organs that are in the high-dose range and not included in this study, IMRT reduces cancer risk because of the higher conformality relative to 3D-CRT. Moreover, it should be noted that the study of Xiang et al. . Even though a simple analytical model for a fast 3D assessment of out-of-field doses has been proposed for photon radiotherapy , the DVH5In this study we demonstrated the use of a validated Monte Carlo framework calculating the complete dose equivalent per organ, including the therapeutic and imaging procedures. We reported on the complete patient exposure during paediatric brain cancer treatment, showing a significant contribution from imaging to the out-of-field dose equivalent per organ when proton therapy is used, due to the lower dose equivalent from proton therapy compared to photon therapy techniques. For the specific out-of-field organs studied, it was shown that proton therapy decreases the out-of-field doses and associated risk for secondary cancer.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MD-H: dose and risk calculations, data analysis, writing. GB: imaging dose simulations, writing. US: treatment-planning, risk calculations, data analysis, writing. CB: treatment planning, imaging protocols, writing-review. NV: proton simulations, writing-reviewing. JE: Monte Carlo geometry coding. JW: proton simulations. FSt: photon simulations. FSu: photon simulations and data analysis. RN: data analysis. JD: methodology, writing-review. FV: data analysis. SR: data analysis. MR: photon simulations, data processing. AS: CT scanner geometry, NJ: epidemiological analysis, writing-review. BT: clinical analysis, writing-review. IT-C: project coordinator, writing-review. LB: Monte Carlo simulations, conceptualization, supervision, writing, writing-review, editing. All authors contributed to the article and approved the submitted version.


The therapeutic potential of short interfering RNA (siRNA) to treat many diseases that are incurable with traditional preparations is limited by the extensive metabolism of serum nucleases, low permeability through biological membrane barriers because of a negative charge, and endosomal trapping. Effective delivery vectors are required to overcome these challenges without causing unwanted side effects. Here, we present a relatively simple synthetic protocol to obtain positively charged gold nanoparticles (AuNPs) with narrow size distribution and the surface modified with Tat-related cell-penetrating peptide. The AuNPs were characterized using TEM and the localized surface plasmon resonance technique. The synthesized AuNPs showed low toxicity in experiments in vitro and were able to effectively form complexes with double-stranded siRNA. The obtained delivery vehicles were used for intracellular delivery of siRNA in an ARPE-19 cell line transfected with secreted embryonic alkaline phosphatase (SEAP). The delivered oligonucleotide remained intact and caused a significant knockdown effect on SEAP cell production. The developed material could be useful for delivery of negatively charged macromolecules, such as antisense oligonucleotides and various RNAs, particularly for retinal pigment epithelial cell drug delivery. Small interfering RNA (siRNA) has shown great promise as a tool for targeted gene silencing, making it a promising direction for the development of novel small-molecule-based therapeutics . In 2018Previous reports on improving siRNA delivery were focused on the chemical stabilization of siRNA ,9. DespiMost CPPs are short cationic peptides (6–30 amino acid residues) known for facilitating membrane translocation and/or nuclear localization . They exUtilizing nanoparticles for siRNA delivery allows for its multimerization, and such delivery vectors can be designed in a way to provide multifunctionality and, hence, enhanced delivery. Gold nanoparticles (AuNPs) have become a popular choice for core nanoparticle scaffolds due to their ability to be easily detected and quantified, as well as because of their potential biocompatibility resulting from their chemical stability. These attributes have led to their extensive use in diagnostic and therapeutic areas of research ,40,41,42Many such constructs employ a layer-by-layer approach to further coat the attached siRNA layer with PEI ,57, charTo avoid a multi-component, multi-step, and multi-layer approach but to maintain multifunctionality, a new method was devised. Here, we describe a simple, reproducible one-step method for the synthesis of positively charged AuNPs coated with cysteamine and displaying a cationic CPP, a modification of the Tat48-60 sequence. This nanoconjugate demonstrates a good ability to complex molecules of nucleic acid nature and low cytotoxicity. The method was employed for the construction of siRNA/AuNP complexes and further transfection of ARPE-19 cells that produce the reporter molecule, secreted alkaline phosphatase (SEAP). Our results show that Tat-siRNA-modified AuNPs could serve as vehicles for gene therapy via intracellular delivery without the loss of biological activity. The developed material can be a useful tool in drug delivery of negatively charged macromolecules such as antisense oligonucleotides, different RNAs, etc.The proposed method involves synthesizing gold clusters, which are stabilized by an alkanethiol monolayer in a two-phase aqueous-organic environment using the modified Brust–Schiffrin method . It emplThe stability of CPP-AuNP complexes with nucleic acids was studied by electrophoresis in 15% polyacrylamide gel . We inveThe next series of experiments was devoted to the investigation of the influence of CPP-AuNPs on SEAP secretion: it was demonstrated that the use of developed CPP-AuNPs increased SEAP secretion . The incThe sequence 1795 (5′-UGA CAA CGG GCA ACA ACU CdTdT-3′) was found to be effective at a dose of 0.5 μg when complexed with CPP-AuNPs  but showFirst, we tested the toxicity of CPP from 2 to 45 μM (0.6−13.2 μg), performing the assay 24 h after transfection in a serum-free medium. We found that the peptide was non-cytotoxic up to 15 μM (4.4 μg per well) and exhibited 50% cytotoxicity at 30 μM (8.8 μg per well).Naked siRNA did not show any significant cytotoxicity. The 48-h cytotoxicity levels of 60% and 22% were observed under using Lipofectamine 2000 for transfection of 0.7 μg of siRNA , 2.5 μL per well, when the transfection was performed in a serum-free medium and a serum-containing medium, respectively. The results of cell viability testing 48 h post-transfection in a serum-free medium (Opti-MEM) and in a serum-containing medium (DMEM/F-12) are presented in During the development of the synthetic procedure of peptide-modified AuNPs, one of the most important parameters to optimize was the ratio between cysteamine and CPP in order to adjust the attachment of two to three CPP molecules per nanoparticle. Stable complexes can be obtained at a molar ratio of cysteamine to cell-penetrating peptides, corresponding to 40/1. The TEM images showed gold nanospheres with diameters of 22.1, 20.9, and 13.4 in three different batches with corresponding SDs of 4.5, 3.5, and 2.0 nm. The UV–Vis spectra of CPP-AuNPs showed a localized surface plasmon resonance band at 520 nm, characteristic of AuNPs b. The abw/w), as indicated by the lack of free oligonucleotide seen in gel electrophoresis  exhibited activity when combined with CPP-AuNPs at a concentration of 0.5 μg , performing the assay 24 h post-transfection in a serum-free medium. Interestingly, an LD50 corresponding to a concentration of 30 µM was demonstrated, while a concentration of 15 µM was found to be practically non-toxic.3O4 nanostructures could be a possible solution [However, results from the MTT assay conducted 48 h after transfection showed significantly lower levels of cytotoxicity for the peptide ; it did solution .Meade and Dowdy et al. have argued that non-covalent siRNA/CPP complexes are the best approach, since the positive charge of the CPP may be neutralized by covalently attached siRNA . FurtherNotably, the functionalization principle of the developed CPP-AuNP with nucleic acids is primarily based on electrostatic interactions. This makes the principle quite versatile for the creation of agents of other types, for example, plasmid-functionalized vectors. However, the development of each novel agent will require optimization of parameters, e.g., empirical identification of the proper ratio of the particle/plasmid amounts, along with the experimental verification of their operation.Future prospects of the present research include extensive studies of the intracellular fate of silencing agents obtained according to the proposed method. At this point, the observed gene silencing mediated by the developed siRNA/CPP-AuNPs suggests that at least some of the agents escape from endosomes. Meanwhile, many research papers, which address the post-uptake intracellular localization of functionalized AuNPs, demonstrate the influence of a multitude of factors, such as coating, size, shape, potential, and many others, on the particle fate after delivery, and no universal scheme was revealed ,70,71. TAs the present research does not include a profound examination of the intracellular fate of the developed siRNA/CPP-AuNPs complexes, there is a possibility that they may be taken up by lysosomes, where they could be either degraded or directed to other organelles. Such events may cause further disruptions in the cellular redox system and potentially result in alterations in the intracellular pH.The findings of the present research can be used for the development of methods for siRNA delivery in vitro. Although the CPP-AuNP-based delivery methods have the potential to provide valuable insights for novel therapeutic approaches ,73,74, i2O, EDT, thioanisol, and TFA was applied to the resin at room temperature for 2 h in the ratio of 1.5:1:1:1:10. Coupling reagents, derivatives of amino acids, and resins used in this work were procured from GL Biochem Ltd., Shanghai, China. The crude peptide was precipitated from the cleavage mix with ice-cold diethyl ether, filtered, and dried. Purification was achieved through reverse phase-high performance liquid chromatography (RP-HPLC) using a Supelco AscenticTM C18 semi-preparative column with dimensions of 10 × 150 mm and particle size of 5 μm. HPLC traces of crude peptide as well as the profile of elution of peptide after purification were very similar to those published in supplementary materials of Ref. [The Tat-linked CPP (H-CGRKKRWWPQRWWRWWRPPQ-OH) was generated based on the methods described in Ref.  using an of Ref. . After t2 × 6H2O, and a pH of 7.5.All siRNAs, including the model double-stranded oligodeoxyribonucleotide, were synthesized using an ASM-800 DNA/RNA synthesizer . Each strand was synthesized separately by following the standard RNA synthesis procedures and utilizing TOM-protected monomers . The cleavage and deprotection protocols specified by the supplier were followed. Purification of synthesized oligonucleotides was performed by IE HPLC followed by desalting by PolyPak II cartridges (Glen Research) according to standard protocols. The siRNA concentration was determined by measuring the absorbance of each strand at 260 nm and using conversion factor of 1 OD being equal to 40 μg/mL of single-stranded RNA. The siRNA strands were immediately annealed before conducting biological experiments, using Dharmacon siRNA buffer consisting of 20 mM KCl, 6 mM HEPES, 0.2 mM MgCl2O was added while stirring at room temperature and incubated for 5 min. A solution containing 0.02 mg of NaBH4  in 0.5 mL of deionized water was added dropwise over a 20-minute period. The mixture was then stirred vigorously and left overnight. The mixture was then moved to a dialysis tube  and dialyzed against dH2O for 48 h. The final product, a suspension of CPP-AuNPs, was stored in a glass container at room temperature. Additional CPP-AuNPs were produced using the same method, with only the ratio between the CPP and cysteamine being altered. The CPP-AuNPs concentration was estimated by assuming complete reduction of gold chloride present in the reaction mixture. This calculation was based on the amount of gold chloride used, which was 5 mg, and the total volume of the reaction solution, which was 9.5 mL. This resulted in a CPP-AuNPs concentration of 0.5 mg/mL gold. The data on the size of the particles (measured to be 20 nm) was utilized to calculate the number of gold atoms per particle and the molar concentration of the solution through application of equations from Ref. [The cell-penetrating peptide-modified gold nanoparticles (CPP-AuNPs), with a 40:1 (mol/mol) ratio of the surface stabilizing agent (cysteamine) to CPP, were prepared as follows: 5 mg (14 μmol) of gold (III) chloride trihydrate  was dissolved in 5 mL of deionized water. After that, a solution containing 1.55 mg (0.53 μmol) of the peptide and 2.4 mg of cysteamine in 4 mL of dHrom Ref. .The siRNA-AuNP complexes were prepared by adding double-stranded siRNA in RNase-free water to CPP-AuNP in different equivalents, and their stability was evaluated using electrophoresis with a double-stranded oligodeoxyribonucleotide as an siRNA model (as described in the subsequent section).CPP-AuNPs were characterized with JEOL 1200-EX II transmission electron microscope , on carbon-coated copper grids, with 100–200 k magnifications. The particle diameters were measured manually using Scion Image 4.0.2 software .The optical properties of both the CPP-AuNPs and siRNA/CPP-AuNP complexes were measured using absorption spectroscopy, covering a wavelength range from 200 to 800 nm. All the measurements were performed using a Cary 100 UV–Vis spectrophotometer .The presence of free CPP in the reaction mixture was assessed through RP-HPLC analysis after the completion of CPP-modified AuNP formation. The CPP-AuNPs were precipitated by adding 100 μL of 2 M HCl and then centrifuged. The amount of unattached peptide was analyzed by RP-HPLC, following the same procedure for peptide synthesis, and compared to a calibration curve generated for peptide concentrations ranging from 0.05 to 1.0 mg/mL.A synthetic double-stranded oligodeoxynucleotide, which possessed the identical sequence as the functional siRNA, was used as a representative molecule to evaluate the stability of the siRNA/CPP-AuNP complexes. The complex stability at different oligonucleotide:peptide mass ratios was tested with 15% PAGE. Stable complexes could be indicated by the lack of unbound oligonucleotide in the electrophoresis gel.Human ARPE-19 retinal pigment epithelial cells were incubated overnight in 100 mm dishes . Then, the cells were exposed to a mixture of 7 μg pCMV-SEAP2/neo reporter plasmid and 25 kDa branched polyethyleneimine (PEI25) with a charge ratio of +4. G418 was added to culture medium at a concentration of 0.8 mg/mL as a selective agent. After the colonies formed, they were harvested and grown in 48 wells plated with medium containing 0.6 mg/mL G418. The expression of alkaline phosphatase was determined through a chemiluminescent assay  and multiple colonies exhibiting varying levels of expression and normal growth were chosen for further experiments.2. To maintain growth, the cells were subcultured weekly.Engineered SEAP-ARPE-19 cells were cultured in nutrient-rich media consisting of DMEM/F-12, fetal bovine serum, L-glutamine, penicillin, streptomycin, and G418. Incubation was performed at a temperature of 37 °C with 7% COFor further transfection, the cells were seeded in 96-well plates and grown in 100 μL of growth medium. After 24 h, the transfection mixtures were added and the cells were either washed with PBS or grown in fresh medium. The impact of CPP-AuNPs on SEAP expression was monitored over time at 6, 24, and 48 h post-transfection, and the effect on cell viability was studied 24 or 48 h post-transfection. Cell proliferation was also studied by counting the number of transfected and non-transfected cells at 24 and 48 h post-transfection using erythrosine dye and a manual cell counter.Great EscAPe Chemiluminescence Kit 2.0 (Clontech) was used to measure the SEAP levels in cell supernatants. The assay was performed according to the protocol recommended by the manufacturer. Briefly, a 60-microliter aliquot was taken from each sample of culture medium. The samples were centrifuged at 12,000 rpm for 1 min and the supernatants were frozen at −20 °C for future analysis. Before assaying, samples were equilibrated to room temperature and then were diluted 20-fold in conditioned growth medium and 15 μL of each was transferred to a 96-well plate . Then, 45 μL of the kit 1× Dilution Buffer was added to each sample. The plate was sealed and incubated at 65 °C for 30 min, after that samples were cooled on ice for 2–3 min and equilibrated to room temperature. After that, the samples were incubated for 60 min with SEAP substrate solution (60 μL). Finally, the SEAP signal was detected and recorded using either a Microbeta 1450 luminometer/liquid scintillation counter  or VarioSkan Flash multimode reader .2. Then, the medium was removed and 100 μL of DMSO was added to each well to dissolve the formazan crystals produced by living cells. VarioSkan Flash multimode reader was used for absorbance measurements at a wavelength of 570 nm. To put in evidence that AuNPs did not affect the MTT reading, cells transfected with high concentrations of CPP-AuNPs were tested via modified assay procedure where only culture medium was added to cells instead of the described mixture.MTT assay was used to investigate the impact of transfection on cell viability. Solution of MTT in PBS (5 mg/mL) was previously prepared and filtered with a 0.2 μm filter. After transfection and following incubation (during 24 or 48 h), cells were rinsed with PBS and a mixture of 10 parts of DMEM/F-12 and 1 part of the MTT solution was added to each well, including control wells without cells and wells with non-transfected cells. Incubation was performed for 2 h at a temperature of 37 °C with 7% COt-tests to determine whether the values were to be considered statistically significant . The following marks were used to reflect statistical significance: ns—p > 0.05; *—p ≤ 0.05; **—p ≤ 0.01; ***—p ≤ 0.001; ****—p ≤ 0.0001.The results of each group are expressed in mean ± standard deviation (SD; n = 5 for A straightforward method to synthesize positively charged AuNPs with surface modification by cell-penetrating peptides, and without any additional steps of polymer surface modifications, has been developed. The synthesized peptide-modified AuNPs were thoroughly characterized by the TEM and SPR techniques and demonstrated a good ability to complex molecules of nucleic acid nature as well as a capacity to serve as vehicles for gene therapy via intracellular delivery without loss of siRNA biological activity. Importantly, the obtained nanoconjugates demonstrated low cytotoxicity. In conclusion, the method has the potential to be extended for the creation of novel sensing surfaces  and func


Glioma is a common primary central nervous system tumor with complex pathogenesis. DNA damage and repair (DDR) is widely involved in regulating cell proliferation and tumorigenesis by correcting and repairing DNA damage mechanisms. Recent studies have reported the following properties in cancer cells in glioma, increased DNA damage and reduced DNA repair capacity. However, the relationship between glioma and DDR-related genes was unclear.DDR-related risk score model was built. The validity of this model was validated in detail through the Kaplan-Meier survival analysis, tumor mutational burden (TMB) analysis, immune cell infiltration, sensitivity to treatment regimens. Moreover, the model’s adaptability was validated in different glioma data cohorts and different glioma subgroups. To further investigate the molecular mechanism of one of DDR-related gene (NUDT1) in glioma, U251 cell was used for the knockdown experiment, followed by MTT, wound healing and transwell analysis.In vitro mechanism study showed that knockdown of NUDT1 reduced tumorigenesis. Furthermore, knockdown of NUDT1 remarkably reduced the expression level of HIF-1α.Ten prognostic-related DDR-related signature genes were obtained, including EID3, MGMT, YWHAG, PMS1, SHPRH, HUS1, NUDT1, GADD45G, APEX1 and FAM175A. The RT-qPCR results suggested that the latter five genes were highly expressed in glioma patients. Interestingly, high TMB score had longer survival. In high-risk score groups, reduced immune cell infiltration in the tumor microenvironment lead to poorer patient outcomes. Sensitivity to treatment regimens analysis indicated that low-risk score groups were more sensitive to chemotherapeutics. Moreover, the risk score model had a good prediction effect on different glioma datasets and different glioma subgroups. Ten prognostic-related DDR-related signature genes were obtained, including EID3, MGMT, YWHAG, PMS1, SHPRH, HUS1, NUDT1, GADD45G, APEX1 and FAM175A.In high-risk score groups, reduced immune cell infiltration in the tumor microenvironment leads to poorer patient outcomes.The risk score model had a good prediction effect on different glioma datasets and different glioma subgroups.Knockdown of NUDT1 reduced tumorigenesis of glioma and remarkably reduced the expression level of HIF-1α.DDR-related risk score model built-in this work has good predictive performance for glioma. RecentlDNA damage and repair (DDR) is a crucial mechanism for correcting and repairing DNA damage, which can timely inhibit cell senescence and carcinogenesis . DDR conFurthermore, it is suggested that tumors with deleterious DDR mutations were more sensitive to platinum-based therapy . The alkDDR alterations are related to clinical and molecular characteristics of glioma. Overexpression of midkine (MDK), a key transcriptional factor in DDR pathways can remodel the glioma immunosuppressive microenvironment by promoting M2 polarization of microglia . There a2.2.1.2(x + 1) processing. Three datasets were downloaded from the Chinese Glioma Genome Atlas (CGGA) database as validation sets [Gene expression data were downloaded from The Cancer Genome Atlas (TCGA) database (involving RNA-seq expression profiling). TCGA database was used as the training set after logion sets . Totallyion sets . Among t2.2.2 fold change (FC)| > 2 and false discovery rate (FDR) < 0.05 was the screening condition of DEGs [In the TCGA cohort, DEGs were identified by the limma. |log of DEGs . DDR-rel of DEGs . The risAccording to the median risk score, patients were divided into the high-risk score and low-risk score groups. Survival analysis was performed using the survminer (R package) to analyze overall survival (OS) in high- and low-risk score groups . The sur2.3.http://research-pub.gene.com/imvigor210corebiologies. Raw count data were converted to transcripts per kilobase million. The CIBERSORT algorithm was used to estimate the infiltrated immune cell component using gene expression signatures.Expression profiles of immune checkpoint-blockaded genes and associated clinical information were downloaded from public databases. Finally, an immunotherapy cohort (IMvigor210) was included: advanced urothelial carcinoma treated with the anti-PD-L1 antibody atezolizumab. Full expression data are available from the URL of 2.4.All independent prognostic parameters and associated clinical parameters were constructed with stepwise Cox regression models to construct prognostic nomogram to predict 1-, 2-, and 3-year OS for glioma patients. A nomogram calibration curve was drawn to compare predicted versus observed OS.2.5.The maftools was utilized to calculate the TMB score. Tumor samples were divided into high- and low-TMB score groups based on the median value of TMB score . Surviva2.6.The ssGSEA algorithm was chosen to quantify the relative abundance of each cell infiltrate in the LAML immune microenvironment . The gen2.7.calcPhenotype algorithm from oncoPredict (R package) was used to evaluate drug IC50 values for each sample in the training set [Based on the Genomics Drug Sensitivity of Cancer Database (GDSC), the ning set . To asse2.8.P value < 0.05 was regarded as statistically significant.To gain an in-depth understanding of the pathogenic mechanism of glioma, the patients were divided into different subgroups based on different clinic-pathological characteristics, including age (≥ 60 and < 60), gender, etc. Fisher’s exact test was utilized to compare the differences in different clinical indicators between high- and low-risk groups. 2.9.Table S1. RT-qPCR was performed by using ABI 7500 with SuperReal PreMix Plus Kit . The 2−ΔΔCt method was utilized to calculate the relative expression level of the gene. ACTB was used as internal reference. This study was approved by the Ethics Committee of West China Hospital. All patients have provided informed consent.Firstly, RNA was extracted from tissues by using TRIzol™ Reagent . The RNA was then reversed into cDNA by using FastKing RT Kit . All gene primers used are displayed in 2.10.2 at 37 °C incubator.To further investigate the molecular mechanism of one of DDR-related gene in the risk score model, U251 cell was used for knockdown experiment, followed by MTT, wound healing and transwell analysis. U251 cell was cultured in medium containing 10% of fetal bovine serum and 5% of CO2.11.4 cells/mL. 100 uL of cell suspension was added to each well of the 96-well plate and cultured for 1, 2 and 3 days. 10uL of CCK-8 solution was added to the 96-well cell culture plate and incubated for 4 h. The absorbance at 450 nm was measured with a microplate reader.The cells were digested by trypsin, counted, and adjusted to concentration of 1x102.12.5/mL in a serum-free medium containing 0.1% of BSA. 500 uL of complete medium was added into 24-well plate. 100 uL of cell suspension was added in Transwell chamber. The cells were transferred to a 24-well plate containing the complete culture medium and incubated for 24h in the cell incubator. After removing the upper chamber medium, the cells were wiped with the moistened cotton swab. The bottom of Transwell chamber was immersed in 10% of the methanol solution to fix the cells for 30 s and transferred to pure water. After washing off the methanol, the bottom of Transwell chamber was immersed in crystal violet dye for dyeing for 2 min, and cleaned with pure water until the background was clear. Microscope photography was performed.Before the experiment, the cells were starved in a serum-free medium for 12h. The cells were digested with trypsin and centrifuged at 1000 rpm for 5 min. After removing the supernatant, the cells were washed with PBS. After the supernatant was discarded, the cells were suspended in a serum-free medium containing 0.1% of BSA for cell count. The cell density was adjusted to 2x102.13.All statistics were performed using R software. The Wilcox test was used to screen for infiltrating immune cells with statistically different. Kaplan-Meier curves were drawn. Log-rank was utilized to test for significant differences in OS between groups. Univariate and multivariate Cox proportional hazards regression analyses were also performed to understand the relationship between risk score and OS. ROC analysis was used to evaluate the sensitivity and specificity of the risk score. The area under the ROC curve (AUC) was an indicator of the accuracy of the prognosis. In the RT-qPCR, paired t-test was used for statistical analysis.3.3.1.http://ualcan.path.uab.edu/)   . Signifi3.2.To access the efficiency of the risk score model, glioma patients were divided into two groups according to the median risk score . The rel3.3.To test the stability of the prognostic risk score model, the mRNA-array_301 cohort, mRNAseq_325 cohort, and mRNAseq_693 cohort were selected to perform the stability analysis. In the mRNA-array_301 cohort, patients in the low-risk score groups had a longer survival time . AUC was3.4.Univariate and multivariate Cox analyses of variables were employed to determine whether the risk score was an independent prognostic factor for OS. Risk score was significantly associated with OS in univariate Cox analysis . After a3.5.To explore the applicability of the constructed prognostic model in different glioma populations, subgroup analyses were performed. Patients in the high-risk score group had a worse prognosis than those in the low-risk score group in all subgroups .3.6.p = 0.011)  . Patient= 0.011) . To furt= 0.011) .3.7.To analyze the relationship of the risk score and the TIME, we evaluated the status of 23 immune cell infiltrations in the TCGA_ glioma tumor sample dataset. Infiltration degree of most immune cells in the high-risk score group was significantly higher than that in the low-risk score group, such as activated dendritic cells, central Memory CD8+ T cells, central memory CD4+ T cells, etc. . Immune 3.8.Identification of new predictive markers is critical for effective immunotherapy. We used the IMvigor210 cohort, which uses anti-PD-L1 immunotherapy, to explore whether DDR-related signature genes can predict the benefit of immunotherapy. Patients with high-risk score had a worse prognosis than patients with low-risk score . The pro3.9.In the GDSC database, many clinically functional genes are targets of anticancer drugs. To further assess the effects of the risk score on drug response, the spearman correlation analysis of the IC50 value of the drug and the risk score was performed. The IC50 value of the drug was significantly positively related to the risk score . Interes3.10.In view of analysis results and related literature reports, one of DDR related gene, NUDT1, was used for further molecular mechanism analysis. From the CCK-8 experiment, we found that NUDT1 promoted tumor cell proliferation at 24, 48 and 72h after transfection . Cell mi4.Recent developments in the molecular mechanisms of the cell cycle have led to renewing interest in gaining a deeper understanding of glioma . FurtherIn this study, we identified 10 DDR prognostic signature genes and constructed a risk score model. Among these, EID3, MGMT, PMS1, and NUDT1 were significantly related to the prognosis and survival of glioma patients, and the high-risk score group had a significantly shorter survival time. EID3, belonging to a member of the IED family, is involved in regulating tumor development in different cancer types ,31. EID3In vitro experiments, we further investigated the molecular mechanism of NUDT1. The result showed that knockdown of NUDT1 reduced tumorigenesis. Furthermore, knockdown of NUDT1 significantly decreased the expression levels of HIF-1α. In solid tumours, hypoxia is common and may alter the response of tumour cells to NUDT1 inhibitors, which have an impact on redox signaling [The above literature has fully demonstrated that the regulation of DDR-related genes on glioma progression. We adopted a comprehensive analysis approach to fully validate the validity of the risk score model for prognosis prediction in different cohorts. ignaling . It is sTMB analysis is important for an in-depth understanding of the pathogenesis of tumors. On the one hand, TMB driver gene mutations can result in tumorigenesis. On the other hand, a large number of somatic mutations can generate neoantigens, which can activate CD8+ cytotoxic T cells, so as to exert T cell-mediated anti-tumor effect . In thisTo further evaluate the validity of the risk score model, we performed immunotherapy analysis and chemotherapeutic drug sensitivity analysis in different risk score groups. Excitingly, the low-risk score group was more sensitive both in response to immunotherapy and chemotherapy, which may be one of the reasons for the better prognosis. However, this work also has limitations. This paper only considers the impact of DDR-related genes on glioma from the transcriptional level, and some other factors, such as the environment, diet, genetics, gene modification, etc., are not considered.5.DDR-related risk score model built-in this work has good predictive performance for glioma.Click here for additional data file.